Ligand supplementation as a method to increase soluble heterologous protein production

Abstract

Ligand interactions are central to enzyme or receptor function, constituting a cornerstone in biochemistry and pharmacology. Here we discuss a ligand application that can be exploited to significantly increase the proportion of recombinant protein expressed in soluble form, by including ligands during the culture. Provided that a sufficiently soluble, cell-permeable and avid ligand is available, one can use it to stabilize nascently synthesized proteins, and in this manner promote solubility and prevent aggregation. To our knowledge, this concept has not been explored systematically and we provide here the first data on ligand supplementation in expression experiments across a whole human protein family: the short-chain dehydrogenases/reductases (SDR). We identified glycerrhitinic acid and its hemisuccinate ester, carbenoxolone (CBX), as ligands with variable affinities ranging from low nanomolar to micromolar binding constants against several SDRs. CBX was utilized as a culture additive in Escherichia coli expression systems against a total of approximately 500 constructs derived from 65 SDR targets, and significantly higher levels of soluble protein were obtained for more than four distinct targets. One of these, the glucocorticoid-activating enzyme type 1 11β-hydroxysteroid dehydrogenase (11β-HSD1), was solubly expressed only at a very low level (<10 µg/l culture) in the absence of ligand; however, soluble expression could be enhanced to mg/l levels by inclusion of CBX or other inhibitors. Other compounds with different chemical scaffolds were used against 11β-HSD1 in equivalent expression experiments yielding similar results. Taken together, if suitable ligands for a given protein are available, this approach could be tested quickly and might represent an easy and effective strategy to enhance soluble protein production, suitable for structural and functional characterization studies.

Keywords::

• 11β-hydroxysteroid dehydrogenase
• carbenoxolone
• heterologous expression
• ligand supplementation
• protein production
• structural genomics

Acknowledgements

We thank Stefan Svensson, Bjorn Elleby and Lars Abrahmsen (Biovitrum, Stockholm, Sweden) for collaboration on initial experiments, and Sujata Sharma (SGC Toronto, Canada) for cloning of SDR genes.

We are also indebted to Rolf Thieringer (Merck, Rahway, NJ, USA) and Nigel Vikker (Sterix, Bath, UK) for providing 11β-hydroxysteroid dehydrogenase inhibitors. Preliminary results were presented at the Peptalk meetings (Cambridge Healthtech, MA, USA) in San Diego in 2005 and 2007.

Financial & competing interests disclosure

The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.